scholarly journals A systems view of spliceosomal assembly and branchpoints with iCLIP

2018 ◽  
Author(s):  
Michael Briese ◽  
Nejc Haberman ◽  
Christopher R. Sibley ◽  
Anob M. Chakrabarti ◽  
Zhen Wang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Splicing Factors ◽  
Splice Sites ◽  
Dynamic Nature ◽  
The Core ◽  
Spliceosomal Protein ◽  
Systems View ◽  
Transcriptomic Studies

AbstractStudies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to map human spliceosome engagement with snRNAs and pre-mRNAs. This identified over 50,000 branchpoints (BPs) that have canonical sequence and structural features. Moreover, it revealed 7 binding peaks around BPs and splice sites, each precisely overlapping with binding profiles of specific splicing factors. We show how the binding patterns of these RBPs are affected by the position and strength of BPs. For example, strong or proximally located BPs preferentially bind SF3 rather than U2AF complex. Notably, these effects are partly neutralized during spliceosomal assembly in a way that depends on the core spliceosomal protein PRPF8. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

scholarly journals Structural disruption of exonic stem–loops immediately upstream of the intron regulates mammalian splicing

2020 ◽  
Vol 48 (11) ◽  
pp. 6294-6309 ◽  
Author(s):  
Kaushik Saha ◽  
Whitney England ◽  
Mike Minh Fernandez ◽  
Tapan Biswas ◽  
Robert C Spitale ◽  
...  
Keyword(s):  
Structural Information ◽  
Structural Features ◽  
Splicing Factors ◽  
Splice Sites ◽  
Exonic Splicing Enhancer ◽  
Stem Loop ◽  
The Core ◽  
Retained Introns ◽  
Splicing Enhancer ◽  
Structural Disruption

Abstract Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem–loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

scholarly journals Structural disruption of exonic stem-loops immediately upstream of the intron regulates mammalian splicing

2018 ◽  
Author(s):  
Kaushik Saha ◽  
Whitney England ◽  
Mike Minh Fernandez ◽  
Tapan Biswas ◽  
Robert C. Spitale ◽  
...  
Keyword(s):  
Structural Information ◽  
Structural Features ◽  
Splicing Factors ◽  
Splice Sites ◽  
Exonic Splicing Enhancer ◽  
Stem Loop ◽  
The Core ◽  
Retained Introns ◽  
Splicing Enhancer ◽  
Structural Disruption

ABSTRACTRecognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

scholarly journals Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Anastasiia Samsonova ◽  
Krystel El Hage ◽  
Bénédicte Desforges ◽  
Vandana Joshi ◽  
Marie-Jeanne Clément ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Cold Shock ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Binding Protein ◽  
Large Set ◽  
Core Component ◽  
The Core ◽  
Cold Shock Domain ◽  
Global Translation

AbstractThe RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved β-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.

scholarly journals The Sub-Nuclear Localization of RNA-Binding Proteins in KSHV-Infected Cells

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1958
Author(s):  
Ella Alkalay ◽  
Chen Gam Ze Letova Refael ◽  
Irit Shoval ◽  
Noa Kinor ◽  
Ronit Sarid ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Viral Infections ◽  
Rna Binding Proteins ◽  
Nuclear Periphery ◽  
Lytic Cycle ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Viral Rnas ◽  
Infected Cells

RNA-binding proteins, particularly splicing factors, localize to sub-nuclear domains termed nuclear speckles. During certain viral infections, as the nucleus fills up with replicating virus compartments, host cell chromatin distribution changes, ending up condensed at the nuclear periphery. In this study we wished to determine the fate of nucleoplasmic RNA-binding proteins and nuclear speckles during the lytic cycle of the Kaposi’s sarcoma associated herpesvirus (KSHV). We found that nuclear speckles became fewer and dramatically larger, localizing at the nuclear periphery, adjacent to the marginalized chromatin. Enlarged nuclear speckles contained splicing factors, whereas other proteins were nucleoplasmically dispersed. Polyadenylated RNA, typically found in nuclear speckles under regular conditions, was also found in foci separated from nuclear speckles in infected cells. Poly(A) foci did not contain lncRNAs known to colocalize with nuclear speckles but contained the poly(A)-binding protein PABPN1. Examination of the localization of spliced viral RNAs revealed that some spliced transcripts could be detected within the nuclear speckles. Since splicing is required for the maturation of certain KSHV transcripts, we suggest that the infected cell does not dismantle nuclear speckles but rearranges their components at the nuclear periphery to possibly serve in splicing and transport of viral RNAs into the cytoplasm.

scholarly journals The Effect of Single CpG Demethylation on the Pattern of DNA-Protein Binding

2019 ◽  
Vol 20 (4) ◽  
pp. 914 ◽  
Author(s):  
Barbara Sobiak ◽  
Wiesława Leśniak
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Epidermal Differentiation ◽  
Keratinocyte Differentiation ◽  
Splicing Factors ◽  
Targeted Next Generation Sequencing ◽  
Factors Analysis ◽  
Epidermal Differentiation Complex ◽  
Complex Process ◽  
Next Generation Sequencing Ngs

Epidermal differentiation is a complex process and its regulation may involve epigenetic factors. Analysis of DNA methylation in 20 selected regions within the epidermal differentiation complex (EDC) gene cluster by targeted next-generation sequencing (NGS) detected no or only minor changes in methylation, mostly slight demethylation, occurring during the course of keratinocyte differentiation. However, a single CpG pair within the exon of the PGLYRP3 gene underwent a pronounced demethylation concomitant with an increase in PGLYRP3 expression. We have employed a DNA-affinity precipitation assay (DAPA) and mass spectrometry to examine changes in the composition of proteins that bind to DNA containing either methylated or unmethylated CpG. We found that the unmethylated probe attracted mostly RNA binding proteins, including splicing factors, which suggests that demethylation of this particular CpG may facilitate PGLYRP3 transcription and/or pre-mRNA splicing.

scholarly journals Challenges and perspectives for structural biology of lncRNAs—the example of the Xist lncRNA A-repeats

2019 ◽  
Vol 11 (10) ◽  
pp. 845-859 ◽  
Author(s):  
Alisha N Jones ◽  
Michael Sattler
Keyword(s):  
High Resolution ◽  
Structural Biology ◽  
Recent Progress ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Molecular Features

Abstract Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5′ end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. Here, we discuss recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats. We discuss the experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. We propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

scholarly journals The Drosophila RNA binding protein Nab2 patterns dendritic arbors and axons via the planar cell polarity pathway

2021 ◽  
Author(s):  
Kenneth H. Moberg ◽  
Edwin B. Corgiat ◽  
Sara List ◽  
J. Christopher Rounds ◽  
Dehong Yu ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Planar Cell Polarity ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Axon Growth ◽  
Planar Cell Polarity Pathway ◽  
Translational Repressor ◽  
The Core ◽  
Dendritic Arbors ◽  
Axon Projection

RNA binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RBP is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules, and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RBP Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. Here, we demonstrate that Nab2 restricts branching and projection of larval sensory dendrites via the planar cell polarity pathway, and that this link may provide a conserved mechanism through which Nab2/ZC3H14 modulates projection of both axons and dendrites. Planar cell polarity proteins are enriched in a Nab2-regulated brain proteomic dataset. Complementary genetic data indicate that Nab2 guides dendrite and axon growth through the planar-cell-polarity pathway. Analysis of the core planar cell polarity protein Vang, which is depleted in the Nab2 mutant whole-brain proteome, uncovers selective and dramatic loss of Vang within axon/dendrite-enriched brain neuropil relative to brain regions containing cell bodies. Collectively, these data demonstrate that Nab2 regulates dendritic arbors and axon projection by a planar-cell-polarity-linked mechanism and identify Nab2 as required for accumulation of the core planar cell polarity factor Vang in distal neuronal projections.

scholarly journals Pan-cancer analysis of RNA binding proteins (RBPs) reveals the involvement of the core pre-mRNA splicing and translation machinery in tumorigenesis

2018 ◽  
Author(s):  
Bin Zhang ◽  
Kamesh R. Babu ◽  
Chun You Lim ◽  
Zhi Hao Kwok ◽  
Jia Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Copy Number ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Human Cancer ◽  
Mrna Splicing ◽  
Regulatory Mechanisms ◽  
Translation Machinery ◽  
The Core ◽  
Cancer Types

AbstractRNA binding proteins (RBPs) are key regulators of posttranscriptional processes such as RNA maturation, transport, localization, turnover and translation. Despite their dysregulation in various diseases including cancer, the landscape of RBP expression and regulatory mechanisms in human cancer has not been well characterized. Here, we analyzed mRNA expression of 1487 RBPs in ~6700 clinical samples across 16 human cancer types and found that there were significantly more upregulated RBPs than downregulated ones in tumors when compared to their adjacent normal tissues. Across almost all of the 16 cancer types, 109 RBPs were consistently upregulated (cuRBPs) while only 41 RBPs were consistently downregulated (cdRBPs). Integrating expression with the copy number and DNA methylation data, we found that the overexpression of cuRBPs is largely associated with the amplification of copy number, whereas the downregulation of cdRBPs may be a result of epigenetic silencing mediated by DNA methylation. Furthermore, our results indicated that cuRBPs could work together to promote cancer progression potentially through the involvement of splicing and translation machinery, while cdRBPs might function independently to suppress tumorigenesis. Additionally, we focused on colon cancer and identified several novel potential oncogenic RBPs, such as PABPC1L which might promote cancer development via regulating the core splicing machinery. In summary, we showed distinct expression landscapes, regulatory mechanisms and characteristics of cuRBPs and cdRBPs and implicated several novel RBPs in cancer pathogenesis. Moreover, our results suggest that the involvement of the core pre-mRNA splicing and translation machinery could be critical in tumorigenesis.

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